The feline major histocompatibility complex (MHC) has been studied using molecular techniques as an approach to comparative genome organization of this important gene cluster. The MHC encodes two types of antigen-presenting molecules for T-cell receptors (class I and class II antigens) and controls immune responses to various pathogens and tumors. Analysis of feline DRA and DRB genes by cDNA cloning, polymerase chain reaction amplification, and nucleotide sequencing yielded the following conclusions: (i) Feline DRA and DRB genes have two loci for each gene. (ii) Highly polymorphic residues are located on the antigen binding groove of DRB molecules but not on DRA molecules. (iii) Five major lineages of DRB sequences are revealed by analysis of 83 distinct alleles from 37 domestic cats in eight countries from five continents. (iv) DRB alleles of three exotic feline species, separated from domestic cat 10-15 million years ago, showed close association with some DRB lineages of domestic cat. However, DRB alleles of other species including human, mouse, and dog were clearly different from these feline DRB alleles. These results suggest that the feline DRB allelic lineages are very old and are derived from alleles of common ancestral feline species which existed 10-15 million years ago. (v) Natural selection pressures in favor of polymorphism in antigen recognition sites (positive selection) and against mutational changes in the framework of other regions (negative selection) play an important role in maintaining the patterns of polymorphism of MHC DRB alleles.